Actively Targeting Redox-Responsive Multifunctional Micelles for Synergistic Chemotherapy of Cancer

Stimuli-responsive polymeric micelles decorated with cancer biomarkers represent an optimal choice for drug delivery applications due to their ability to enhance therapeutic efficacy while mitigating adverse side effects. Accordingly, we synthesized a digoxin-modified novel multifunctional redox-responsive disulfide-linked poly(ethylene glycol-b-poly(lactic-co-glycolic acid) copolymer (Bi(Dig–PEG-PLGA)-S2) for the targeted and controlled release of doxorubicin (DOX) in cancer cells. Within the micellar aggregate, the disulfide bond confers redox responsiveness, while the presence of the digoxin moiety acts as a targeting agent and chemosensitizer for DOX. Upon self-assembly in aqueous solution, Bi(Dig–PEG-PLGA)-S2 formed uniformly distributed spherical micelles with a hydrodynamic diameter (Dh) of 58.36 ± 0.78 nm and a zeta potential of −24.71 ± 1.01 mV. The micelles exhibited desirable serum and colloidal stability with a substantial drug loading capacity (DLC) of 6.26% and an encapsulation efficiency (EE) of 83.23%. In addition, the release of DOX demonstrated the redox-responsive behavior of the micelles, with approximately 89.41 ± 6.09 and 79.64 ± 6.68% of DOX diffusing from DOX@Bi(Dig–PEG-PLGA)-S2 in the presence of 10 mM GSH and 0.1 mM H2O2, respectively, over 96 h. Therefore, in HeLa cell lines, DOX@Bi(Dig–PEG-PLGA)-S2 showed enhanced intracellular accumulation and subsequent apoptotic effects, attributed to the targeting ability and chemosensitization potential of digoxin. Hence, these findings underscore the promising characteristics of Bi(Dig–PEG-PLGA)-S2 as a multifunctional drug delivery vehicle for cancer treatment.


INTRODUCTION
Cancer is one of the most challenging and complex diseases to treat and persists as a major global health concern.−3 Chemotherapy is often prescribed as a supportive treatment for various types of malignancies as it efficiently inhibits their proliferation and impedes metastasis.However, the nonspecific distribution of such chemotherapeutic drugs throughout the body leads to serious side effects on healthy cells. 4,5−8 In this regard, drug delivery systems (DDS) utilizing nanoparticles, micelles, gels, liposomes, polymeric prodrugs, and other polymeric functional materials have been developed.These systems offer advantages, such as biocompatibility and versatility in surface modification and facilitate the selective accumulation and controlled release of chemotherapeutic agents in the vicinity of cancer cells. 9,10Moreover, they have the potential to prevent sequestering payloads by the phagocytic system, reduce accumulation in organs like the liver and spleen, and prolong systemic circulation, thereby enhancing anticancer effects. 11olymeric micelles, often made of amphiphilic block copolymers, are the most preferred nanocarriers for delivering hydrophobic chemotherapeutic agents, owing to their hydrophobic core, capable of solubilizing and encapsulating waterinsoluble agents. 12Moreover, the hydrophilic shell serves as a hydration barrier, increasing colloidal stability and prolonging the systemic circulation by avoiding rapid renal excretion and uptake by the reticuloendothelial system (RES).Subsequently, this prolongs the accumulation of the micellar assemblies in tumor tissue through the enhanced permeability retention (EPR) effect. 13Achieving therapeutic drug concentration in tumor tissue is fundamental to counteract the effects of membrane transporters like P-glycoprotein, which are implicated in the efflux of drugs and suboptimal intracellular concentration, resulting in the emergence of multidrug resistance toward the payloads. 14,15Surface functionalization of polymeric micells via the physical or chemical conjugation of different cancer cell biomarkers, such as antibodies, transferrin, folic acid, aptamers, RGD peptides, etc., can enhance the selective cellular uptake of drug-loaded micelles through receptor-mediated endocytosis. 10,16,17Furthermore, the spatiotemporal release of payloads in the intracellular compartment of cancer cells is equally important to elicit a prompt cancer-killing effect.Most micellar aggregates suffer from premature payload release in the systemic circulation before reaching the target tissue, partly due to diffusion and/or biodegradation-triggered release kinetics. 18This premature release leads to undesirable side effects, suboptimal therapeutic outcomes, and cancer recurrence. 19Thus, the incorporation of stimuli-responsive moieties that respond to external or internal stimuli (such as pH, temperature, reduction, oxidation, light, enzyme, etc.) in micellar systems has garnered significant attention in recent decades.−23 Overall, the fabrication of tumor targeting and stimuli-responsive micellar systems holds promise to address the prospect of premature payload release and nonselective biodistribution of anticancer drugs in the body.
Taking this into account, we synthesized a novel digoxinmodified redox-responsive Bi(Digoxin−PEG-PLGA)-S 2 block copolymer, hereafter referred to as Bi(Dig−PEG-PLGA)-S 2 .This copolymer self-assembled into micelles with a PLGA−S− S-PLGA core and digoxin-PEG shell, intended for the targeted delivery of doxorubicin (DOX) to cancer cells.The presence of digoxin as a targeting moiety facilitates selective and sitespecific internalization of DOX-loaded micelles, as digoxin often interacts with Na + /K + ATPase, which is overexpressed on the plasma membrane of cancer cells.Moreover, the use of digoxin has synergistic therapeutic effects owing to its sensitization of cancer cells toward chemodrugs. 24,25Furthermore, a redox-responsive disulfide moiety was incorporated to achieve spatiotemporal release of DOX within cancer cells.−29 Following receptor-mediated endocytosis and intracellular accumulation in cancer cells, the DOX-loaded Bi(Dig−PEG-PLGA)-S 2 micelles (DOX@Bi(Dig−PEG-PLGA)-S 2 ) can swell or disassemble, subsequently releasing DOX due to the oxidation and reduction of the S−S bond, which trigger phase change in the micellar core. 26In this study, fluorescence microscopy, MTT assay, and flow cytometry analysis were used to examine the in vitro cellular uptake and cytotoxic effect of DOX-loaded Bi(HOOC−PEG-PLGA)-S 2 (DOX@Bi(HOOC−PEG-PLGA)-S 2 ) and DOX@Bi(Dig− PEG-PLGA)-S 2 micelles against HeLa cells.The results revealed that DOX@Bi(Dig−PEG-PLGA)-S 2 exhibited superior cellular internalization and cancer cell inhibition compared to DOX@Bi(HOOC−PEG-PLGA)-S 2 .Moreover, the effect of DOX@Bi(Dig−PEG-PLGA)-S 2 on apoptosis and necrosis of HeLa cells was comparable to that of cells treated with free DOX.

Synthesis of HOOC−PEG-PLGA.
The block copolymer HOOC−PEG-PLGA was synthesized by ring-opening copolymerization (ROP) of GA and LA using HOOC-PEG− OH as an initiator and Sn(Oct) 2 as a catalyst (Scheme S1a) following ref.30 with a slight modification.Briefly, 1.25 mmol of HOOC-PEG−OH was placed into a two-neck reaction flask and dried in a vacuum oven at 110 °C for 3 h to eliminate traces of water.The temperature was reduced to 90 °C, and 32.25 mmol of LA and 10.75 mmol of GA (LA:GA ratio = 3) were added under nitrogen protection and maintained for 30 min to remove the residual moisture.Then, 150 μL of Sn (Oct) 2 catalyst was injected into the reaction mixture and continuously stirred at 150 °C for 24 h.Finally, the crude product was purified by dissolving it in a small amount of DCM and precipitating in large excess cold diethyl ether (repeated three times).The residual solvent was completely eliminated from the precipitate by oven drying under a vacuum at 45 °C for 48 h.The target product was stored in a refrigerator at below 10 °C.

Synthesis of Bi(HOOC−PEG-PLGA)-S 2 .
A disulfidelinked HOOC−PEG-PLGA copolymer (Bi(HOOC−PEG-PLGA)-S 2 ) was prepared by coupling reaction in the presence of DMAP and DCC (Scheme S1b). 26,30Briefly, 0.08 mmol of HOOC−PEG-PLGA copolymer and 0.23 mmol of DMAP were dissolved in a anhydrous THF (20 mL) in a two-neck reaction flask.Then, the mixture was stirred at 0 °C under a nitrogen atmosphere until it was completely dissolved.After 30 min, 0.04 mmol of 3,3′-dithiodiproponic acid (disulfide linker) and 0.23 mmol of DCC were added and stirred at 0 °C and room temperature (∼25 °C) for 4 and 72 h, respectively.The resulting product was purified by membrane dialysis with MWCO = 2 kDa against THF for 8 h and by deionized (DI) water for 48 h (exchanged every 6 h).
2.4.Synthesis of Bi(Dig−PEG-PLGA)-S 2 Copolymer.Bi(Dig−PEG-PLGA)-S 2 was synthesized from Bi(HOOC− PEG-PLGA)-S 2 and digoxin in the presence of catalytic amounts of DMAP and DCC (Scheme S1c). 26,30Shortly, 0.08 mmol of Bi(HOOC−PEG-PLGA)-S 2 copolymer and 0.23 mmol of DMAP were dissolved with anhydrous THF (20 mL) in a two-neck reaction flask.Then, the mixture was stirred at 0 °C under a nitrogen atmosphere.After 30 min, 0.2 mmol of digoxin and 0.23 mmol of DCC were added and stirred at 0 °C and room temperature (∼25 °C) for 4 and 72 h, respectively.The resulting Bi(digoxin−PEG-PLGA)-S 2 was purified by membrane dialysis with MWCO = 2 kDa against THF for 24 h and DI water for 48 h (exchanged every 6 h) to remove unbound digoxin, residual DMAP, and DCC.
2.5.Characterization.The chemical structures of the synthesized copolymers were verified by 1 H NMR using a Varian Unity-600 NMR spectrometer (Varian, Inc., CA, USA) at 600 MHz.In addition, the molecular weights of the respective copolymers were determined using the advanced polymer chromatographic (APC) technique using a THF column (ACUITY APC system).For this purpose, 2 mg/mL of HOOC−PEG-PLGA and Bi(HOOC−PEG-PLGA)-S 2 samples were prepared using THF as a solvent, and the molecular weight and polydispersity index (Đ) were measured at 45 °C at a flow rate of 0.8 mL/min using polystyrene as an internal standard for molecular weight calibration.Moreover, the particle size surface charge or zeta (ζ)-potential of the micelles was measured using a Dynamic Light Scattering (DLS) analyzer (Horiba Zeta sizer-100 system, UK) with a scattering angle of 90°at a temperature of 25 °C in triplicate, and the results were expressed as mean ± SD.Field-Emission Scanning Electron Microscopy (FE-SEM, JSM 6500F, JEOL) was also used to assess the morphology and estimate the particle size of the micelles.Herein, a droplet of diluted micelle solution was spin-coated on a silicon substrate and vacuumdried for 24 h, followed by platinum coating for 10 min before FE-SEM image scanning.The absorbance and emission spectra of the different samples prepared during drug loading, critical micelle concentration (CMC) determination, etc., were also determined using UV−vis spectroscopy (JASCO V-730) and photoluminescence spectroscopy (JASCO V-330), respectively.
2.6.Determination of Critical Micelle Concentration.The CMC of the (Bi(Dig−PEG-PLGA)-S 2 ) copolymer in aqueous solution was determined by the pyrene fluorescent probe method using a fluorescence spectrophotometer (JASCO V-330).Briefly, serial concentrations of 1 × 10°, 1 × 10 −1 , 5 × 10 −2 , 1 × 10 −2 , 5 × 10 −3 , 1 × 10 −3 , and 5 × 10 −4 mg/mL of Bi(Dig−PEG-PLGA)-S 2 copolymer solutions were prepared using the dilution method and stored overnight at 10 °C.In the meantime, pyrene was dissolved in acetone to make a stock solution with a concentration of 2 × 10 −4 M. From this stock solution, 20 μL was transferred into seven different amber glass vials, and the acetone was evaporated in the dark for 12 h.The serial concentrations of the different copolymer solutions were transferred to the respective pyrene-containing vials to obtain a final pyrene concentration of 2 × 10 −5 M and then continuously sonicated for 30 min.After stabilizing overnight at room temperature, the fluorescence emission spectra of pyrene were scanned between 350 and 440 nm with an excitation wavelength of 336 nm and an excitation and emission slit width of 2.5 nm.Finally, the CMC of the copolymer was computed by plotting the intensity ratio of the first peak at 372 nm and the third peak at 383 nm (I372/I383) of the pyrene emission spectra against the copolymer concentrations (log C ).The intersection point of the vertical and horizontal tangent lines of the curve through the points of low concentration is considered as the CMC of the copolymer. 26,31.7.Preparation of Blank and DOX-Loaded Micelles.The self-assembly behavior of Bi(HOOC−PEG-PLGA)-S 2 and Bi(Dig−PEG-PLGA)-S 2 into micellar aggregates was investigated based on the reported protocols with minor modifications. 31,32A 5 mg/mL concentration of copolymer in DMSO was prepared, and the probe was sonicated for 5 min by adding water dropwise.Then, it was transferred into 4-fold ultrapure water drop by drop with gentle stirring and proceeded overnight.DMSO was removed by dialysis against DI water for 48 h, during which water was exchanged at 4 h intervals.Finally, the size and surface charge of the micellar solution were estimated via DLS.Similarly, DOX-loaded micelles were prepared by the solvent exchange method with dialysis. 30,33DOX•HCl (4 mg) was dissolved in 2 mL of DMSO and neutralized with 5 μLTEA to obtain a free and hydrophobic DOX solution.It was subsequently mixed with copolymers (40 mg dissolved in 2 mL of DMSO), probesonicated for 5 min, and added dropwise to 15 mL of PBS under vigorous stirring for 36 h.The DMSO and unbound DOX were then removed by membrane dialysis (MWCO = 1 kDa) against DI water for 24 h (water exchanged at every 4 h) (Figure S3).Finally, the DOX-loaded micellar solutions were collected, and the absorbance of DOX was determined at 485 nm via UV−vis spectrophotometer to compute the drugloading capacity (DLC) and encapsulation efficiency (EE) using a pre-established DOX calibration curve (Figure S4) using the following formula Amount of DOX loaded in the micelle Weight of DOX loaded micelle 100 Amount of DOX loaded in the micelle Amount of DOX initially loaded 100

Colloidal and Serum Stability of Micelles.
The colloidal stability of Bi(Dig−PEG-PLGA)-S 2 micelles was investigated using the dilution method.It is based on the dilution of the micellar solution against a large volume of solvent, usually PBS, which represents the volume of the entire systemic circulation. 31Thus, a 5 mg/mL micellar solution was prepared, and the volume was adjusted to 1000 folds by diluting with PBS.Then, the change in hydrodynamic diameter (D h ) as a function of time was measured in triplicate every 24 h for 7 days.In addition, the serum stability of Bi(Dig−PEG-PLGA)-S 2 micelles was assessed by incubating freshly prepared micelles in 50% FBS (used to simulate biological fluid) and monitoring the change in D h of the micelles as a function of time.
2.9.Redox-Responsive Drug Releasing Behavior of Micelles.The in vitro drug-releasing kinetics of DOX@ Bi(Dig−PEG-PLGA)-S 2 micelles were investigated in a simulated cancer redox environment and at normal physiological conditions (PBS and pH 7.4).In brief, a fixed weight of DOX@Bi(Dig−PEG-PLGA)-S 2 micelles in 2 mL of PBS was placed in a dialysis bag (MWCO = 1 kDa) and immersed into 10 mL of release medium (PBS of pH 7.4, 5 mM GSH, 10 mM GSH, and 0.1 mM H 2 O 2 ) in a 20 mL vial.Then, it was placed in an orbital shaker incubator (Yihder Orbital Shaker, LM-420D) by maintaining the temperature at 37 °C with mild agitating at 100 rpm.At a predetermined time, 1, 2, 3, 4, 6, 12, 24, 48, 72, and 96 h, 3 mL of each release medium was ACS Omega collected from the vials for UV−vis measurement of DOX and replenished with an equal volume of new medium.Finally, the amount of DOX released from the micelles was calculated using the pre-established standard calibration curves of free DOX.
2.10.Cellular Uptake of DOX-Loaded Micelles.The cellular trafficking of DOX@Bi(HOOC−PEG-PLGA)-S 2 and DOX@Bi(Dig−PEG-PLGA)-S 2 micelles against HeLa cell lines was monitored using a fluorescence scanning microscope (iRiSTM Digital Cell Imaging System from Logos Biosystem). 30The HeLa cells were seeded in confocal dishes (2 × 10 5 cells/dish) with complete DMEM medium (containing 10% (v/v) FBS and 1% (v/v) streptomycin) and incubated for 24 h at 37 °C and 5% CO 2 in a humidified incubator.The cells were then treated with free DOX, DOX@Bi(HOOC−PEG-PLGA)-S 2 , and DOX@Bi(Dig−PEG-PLGA)-S 2 at equivalent DOX concentrations of 3 μg/mL.At predetermined time points (4 and 12 h), the culture medium was removed, washed three times with PBS, and stained with DAPI (300 nM) for 30 min to stain the nuclei of HeLa cells.Then, fluorescence cellular images were taken to trace the internalization of DOX@Bi(HOOC−PEG-PLGA)-S 2 and DOX@Bi(Dig−PEG-PLGA)-S 2 into the cells.embryonic fibroblast cells of the NIH/Swiss mouse) and cancer cell lines (HeLa cells and human cervical carcinoma cells).The respective cell lines were seeded in 96-well plate at a density of 1 × 10 4 cells/wall in triplicate using complete culture medium (DMEM supplemented with 10% FBS and 1% streptomycin) and incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO 2 .Then, the old medium was replenished with fresh medium containing Bi(Dig−PEG-PLGA)-S 2 at concentrations of 10, 20, 40, 60, 80, 100, and 200 μg/mL and incubated for another 24 h.The cells were washed three times with PBS and treated with MTT solution (1 mg/mL medium in each well).The MTT solution was removed after 4 h of incubation, and 100 μL of DMSO was added to each well to dissolve the formazan crystals, and the absorbance was measured at 570 nm using a microplate reader (ThermoMultiskan FC microplate photometer, USA).The viability of NIH-3T3 and HeLa cells was calculated according to the following formula Absorbance of test cells Absorbance of control cells 100 Similarly, the cell viability of DOX@Bi(HOOC−PEG-PLGA)-S 2 and DOX@Bi(Dig−PEG-PLGA)-S 2 micelles was determined by the same procedure against NIH-3T3 and HeLa cell lines at an equivalent DOX concentration of 0.5−12.5 μg/ mL.

Flow Cytometry Analysis of DOX-Loaded Micelles.
A fluorescent activated cell sorting (FACS) assay was conducted to quantify the number of live, apoptotic, and necrotic cells.HeLa cells were seeded in 6-well plates (1 × 10 6 cells/well) and incubated for 24 h at 37 °C and 5% CO 2 .After rinsing with PBS, the cells were treated with PBS (control group), free DOX, DOX@Bi(HOOC−PEG-PLGA)-S 2 , and DOX@Bi(Dig−PEG-PLGA)-S 2 (5.5 μg/mL) for 12 h.Then, the cells were rinsed with PBS, detached using trypsin, centrifuged (for 5 min at 1200 rpm), and resuspended in 100 μL of annexin V binding buffer (1X).Next, 2 μM of Alexa fluor 488 annexin-V and PI were added and incubated for 20 min at room temperature (in the dark).Finally, the cell suspensions were diluted with annexin-V binding buffer (400 μL), and the live, apoptotic, and necrotic cells were quantified by flow cytometry instrument (FACScan; Becton Dickinson, Heidelberg, Germany).
2.14.Statistical Analysis.The data values are reported as the mean ± SD of triplicate experiments.The statistical significance of the variances between the two groups was determined using a two-tailed student t-test.A p-value <0.05 was considered as statistically significant.

Synthesis and Characterization of Bi(Dig−PEG-PLGA)-S 2
Copolymer.The amphiphilic block copolymer, Bi(Dig−PEG-PLGA)-S 2 , can self-assemble into micellar aggregates with a core−shell structure capable of encapsulating hydrophobic chemotherapeutic agents. 36The synthesis of this copolymer involves the following steps: first, ring opening polymerization (ROP) of GA and LA in the presence of HOOC-PEG−OH as an initiator to form HOOC−PEG-PLGA. Next, two equiv of HOOC−PEG-PLGA were linked via 3,3′-dithiodiproponic acid to form Bi(HOOC−PEG-PLGA)-S 2 .Finally, Bi(HOOC−PEG-PLGA)-S 2 was decorated with digoxin to prepare Bi(Dig−PEG-PLGA)-S 2 through the DCC/DMAP coupling reaction.During these reactions, DPC and DMAP were used to activate the −COOH group to trigger ester bond formation under dry solvent, anhydrous THF. 26,31 1  NMR spectroscopy was used to verify the chemical structures of the synthesized block copolymers.As shown in Figure 1a, the characteristic peak at δ H 4.9 ppm (a) is ascribed to the CH 2 protons of GA, while the signals at δ H 5.3 ppm (b) and 1.5 ppm (c) represent the CH and CH 3 protons of LA, respectively.Additionally, the peak that appeared between δ H 3.3−3.6ppm (d) confirmed the presence of CH 2 protons of PEG, suggesting the successful synthesis of the HOOC−PEG-PLGA copolymer, which is in agreement with a previous report. 37In the case of the the disulfide-linked copolymer, Bi(HOOC−PEG-PLGA)-S 2 , additional peaks δ H 2.9 and δ H 3.1 ppm are representative of S−CH 2 -CH 2 -COOH protons of 3,3′-dithiodiproponic acid, verifying the successful conjugation of HOOC−PEG-PLGA and 3,3′-dithiodiproponic acid to form Bi(HOOC−PEG-PLGA)-S 2 (Figure 1b).When the terminal −COOH moiety of Bi(HOOC−PEG-PLGA)-S 2 was decorated with digoxin, in addition to the aforementioned proton peaks of HOOC−PEG-PLGA and Bi(HOOC−PEG-PLGA)-S 2 , new peaks that are characteristic of digoxin protons appeared in the 1 H NMR spectrum, as shown in Figure 1c.The peaks that appeared between δ H 0.6−1.2ppm indicated the methyl proton of digoxin.Similarly, the peaks between δ H 1.5−2.1 ppm and δ H 6.0 ppm were assigned to the methylene proton of digoxin, signifying the successful chemical conjugation of Bi(HOOC−PEG-PLGA)-S 2 and the targeting moiety, digoxin.To further confirm the synthesis of block copolymers, an advanced polymer chromatography technique was employed, and an average molecular weight (Mn) of 5713 g/mol with a narrow molecular weight distribution (Đ = 1.23) was recorded for HOOC−PEG-PLGA (Figure S1b and Table 1).Furthermore, as two equivalents of HOOC−PEG-PLGA copolymers were covalently linked by 3,3′-dithiodiproponic acid to form Bi(HOOC−PEG-PLGA)-S 2 , the molecular weight of the resulting copolymer is expected to double. 26onsistently, the average molecular weight of Bi(HOOC− PEG-PLGA)-S 2 was found to be 10 976 g/mol with unimodal distribution (Table 1).The APC chromatogram reiterated the relatively lower retention time (faster elution) for Bi(HOOC− PEG-PLGA)-S 2 than the HOOC−PEG-PLGA copolymer, further asserting the efficient synthesis protocols followed and the successful preparation of Bi(HOOC−PEG-PLGA)-S 2 (Figure S1c).

Critical Micelle Concentration of Bi(Dig−PEG-PLGA)-S 2 .
Above the CMC (a certain concentration), amphiphilic block copolymers undergo self-assembly to form well-defined micelles.This spontaneous entanglement of copolymers in aqueous solution is a reversible thermodynamic process, and strongly depends on the CMC and intermolecular interactions within the micelles.A lower CMC value indicates higher stability of the micellar aggregate, particularly at extreme dilution, making the CMC a crucial measure of polymeric micelle stability. 31,38When micelles are administered into the body, their structural integrity may be altered by the large volume of body fluid in the systemic circulation.Micelles with a higher CMC will undergo spontaneous swelling and disassembly to release their payload before reaching the target tissues.Therefore, the CMC determination is of paramount importance in the fabrication of anticancer drug delivery vehicles, specifically micelles.Because of their amphiphilic nature, Bi(HOOC−PEG-PLGA)-S 2 and Bi(Dig−PEG-PLGA)-S 2 spontaneously form micelles in an aqueous solution with a hydrophobic PLGA core (that can encapsulate DOX or pyrene) and a hydrophilic PEG shell that interact with the surrounding medium.Encapsulation of DOX within Bi-(HOOC−PEG-PLGA)-S 2 and Bi(Dig−PEG-PLGA)-S 2 micelles could improve the solubility and minimize systemic toxicity during therapeutic usage. 31In this study, the CMC of the Bi(Dig−PEG-PLGA)-S 2 copolymer was estimated using a fluorescence spectrophotometer, employing pyrene as the hydrophobic core probe.The CMC of the copolymer was found to be 0.014 mg/mL (Figure S2b), indicating that Bi(Dig−PEG-PLGA)-S 2 copolymer-based micelles might be stable at extreme dilution and could have longer circulation times without premature release of its cargo. 26This CMC value is comparable to those reported for similar amphiphilic block copolymer micelles in previous reports: 0.0091 mg/mL 39 and 0,0139 mg/mL. 26,40.3.Particle Size and Surface Charge of the Micelles.The drug delivery potential of polymeric micelles is strongly correlated to their D h or particle size.Nanosized micelles (≤200 nm) often experienced better extravasation potential through the tight lining of blood vessels and better accumulation in tumor tissue through the EPR effect.Micelles that are too small (≤5 nm) or too large (≥500 nm) may be eliminated from the body through the kidney and RES, respectively.Similarly, the surface charge can also dictate the magnitude of interaction between micelles and cellular structures of cancer cells, 41,42 thereby affecting the cellular internalization and cancer-killing effect of payloads.The D h and surface charge of Bi(Dig−PEG-PLGA)-S 2 micelles were determined using DLS.As shown in Table 2 and Figure 2a, the D h of the Bi(Dig−PEG-PLGA)-S 2 micelles was slightly lower than that of the Bi(COOH−PEG-PLGA)-S 2 micelles, with relatively narrow size distributions (Đ ranged from 0.2 to 0.4).The decrease in the size of micelles prepared from Bi(Dig− PEG-PLGA)-S 2 might be partly attributed to the hydrophobic digoxin terminals that induce tight packing and reduce the size of the micelles.43 However, after DOX was loaded in the hydrophobic core of Bi(Dig−PEG-PLGA)-S 2 micelles, a slight increment in size was noted, indicating that the enclosure of DOX in the micelles' core increased its volume.44,45 Furthermore, the SEM image (Figure 2c−e) confirmed the spherical shape of uniformly distributed polymeric micelles.The estimated particle size from SEM images closely matched with the size obtained with DLS measurement, despite the difference in sample preparation.The surface charges (ζpotential) of both blank and DOX@Bi(Dig−PEG-PLGA)-S 2 micelles were also analyzed using DLS, and the absolute value of ζ-potential was ranged between 20 and 35 mV (Table 2).This falls within a desirable range for enhancing cellular internalization via endocytosis and preventing self-aggregation during storage.46−48 Comparing blank micelle to DOX@ Bi(Dig−PEG-PLGA)-S 2 micelles, a slight shift toward a positive direction in ζ-potential (from −24.71 to −20.67) was observed, which might be due to the presence of an -NH 2 group in DOX that could ionize into a positive charge in aqueous medium.49 3.4.Colloidal and Serum Stability of Micelles.The fabrication of stable micelles with prolonged systemic circulation remains a challenging research area in drug delivery science.The efficient accumulation of micelles into the extracellular matrix or cancer niche demands intact and stable aggregates with a longer circulation half-life, ensuring sustained exposure of cells to payloads.However, the structural integrity of polymeric micelles can be compromised by their undesirable interaction with serum proteins and the dilution effect of the systemic circulation.Because the CMC of copolymers can be affected by the large excess volume of the entire plasma fluid in the body, 38,50 Overall, such detrimental interactions result in disassembly of micelles into polymer chains and rapid clearance of micelles from the circulatory system through the RES. 51,52Taking this into account, the colloidal stability of Bi(Dig−PEG-PLGA)-S2 micelles was examined by incubating 5 mg/mL of the micelles in a 1000-fold volume of PBS and 50% FBS, and the change in Dh of the micelles was monitored for seven consecutive days.As shown in Figure 2b, micelles incubated with PBS (pH 7.4) showed insignificant changes in Dh (60.96 ± 7.22 to 83.27 ± 9.45 nm over 7 days) under physiological conditions (pH 7.4 and 37 °C).Conversely, micelles exposed to 50% FBS remained relatively stable for five consecutive days before experiencing a slight increment in size during the sixth and seventh days.Although the interaction of micelles with the proteins in FBS caused quicker micellar swelling (an increase in Dh) compared to that in micelles incubated with PBS alone, the stability period of micelles in 50% FBS was quite promising.This suggests that they may remain intact in the bloodstream and may achieve longer halflife for effective therapeutic outcome.48

Drug Encapsulation Potential of Bi(Dig−PEG-PLGA)-S 2 .
Polymeric micelles should encamp within a short period of time.Hence, polymeric micelles should possess a relatively higher drug loading and encapsulating efficiency.Bi(HOOC−PEG-PLGA)-S2 and Bi(Dig−PEG-PLGA)-S2 are amphiphilic block copolymers capable of self-assembling into micellar nanostructures in aqueous solution; hence, they can encapsulate hydrophobic drugs, such as DOX, predominantly in the core, and to a lesser extent, at the interface between the hydrophobic and hydrophilic segments.This encapsulation involves a weak hydrophobic interaction between DOX and the core as well as the π−π stacking among DOX molecules. 55n this study, DOX was loaded in the hydrophobic core of Bi(HOOC−PEG-PLGA)-S2 and Bi(Dig−PEG-PLGA)-S2 micelles through the dialysis method.The DLC and EE were calculated by measuring the absorbance of DOX-loaded micelles using a UV−vis spectrophotometer and the preestablished calibration curve of DOX (Figure S4).Bi(Dig− PEG-PLGA)-S2 exhibited relatively higher DLC (6.26%) and EE (83.23%) compared to those of Bi(HOOC−PEG-PLGA)-S2 micelles (DLC, 6.01% and EE, 79.19%).This slight variation in DLC between two micellar formulations might be due to the adsorption of DOX molecules on the surface of the micelle due to the favorable π−π interaction with hydrophobic digoxin present in the shell of Bi(Dig−PEG-PLGA)-S2. 53−55 Overall, the DLC and EE of Bi(Dig−PEG-PLGA)-S2 were more acceptable as compared with disulfide-linked redoxresponsive systems designed for DOX delivery. 31,56.6.Redox-Responsive Drug Releasing Behavior.In drug delivery applications, the release kinetics of payloads is crucial for achieving therapeutic concentration in the target cellular compartment and eliciting the intended bioactivity.Hence, stimuli-responsive drug delivery vehicles have become increasingly fabricated to ameliorate the anticancer activities of hydrophobic drugs such as DOX compared with conventional diffusion-triggered release systems.These vehicles offer spatiotemporal release kinetics in and around cancer cells.In this study, the release of DOX from the micelles was investigated under different conditions to mimic normal physiological conditions (PBS, pH 7.4) and cancer environments (5 mM GSH, 10 mM GSH, and 0.1 mM H 2 O 2 ).The intracellular environment in cancer cells differs from that of normal cells (i.e., in cancer cells, the concentration of GSH and H 2 O 2 exceeded by more than 1000 folds as compared to normal cells).This difference could contribute to micellar swelling or disassembly and controlled release of the cargo within cancer cells.−59 As depicted in Figure 3, the release of DOX in reducing (5 mM GSH and 10 mM GSH) and oxidizing environments (0.1 mM H 2 O 2 ), representing the cancer environment occurred much more abruptly than the release profile of DOX in PBS (pH 7.4), a normal physiological environment.The cumulative release of DOX (65.51 ± 8.21%) at higher concentrations of GSH (10 mM) within the first 24 h was significantly higher than the cumulative release of DOX (22.68 ± 5.01%) in the same time period in PBS.At 72 h, approximately 71.74 ± 7.22% and 82.78 ± 4.57% of DOX were released in GSH (5 mM) and GSH (10 mM) releasing media, respectively.After 96 h, nearly all DOX (89.41 ± 6.09%) was evacuated from DOX@Bi(Dig− PEG-PLGA)-S 2 micelles into the surrounding 10 mM GSHcontaining releasing media.Similarly, the cumulative release of DOX in the presence of 0.1 mM H 2 O 2 at 96 h was about 79.64 ± 6.68%.In this circumstance, the amount of DOX released in the cancer redox environment was therapeutically sufficient for inducing necrosis of cancer cells even in the first 24 h.Interestingly, the cumulative release of DOX in PBS release medium was only 37.95 ± 5.08% within 96 h, suggesting minimal drug leakage under normal physiological conditions, thus minimizing undesired side effects on normal cells.The results are in line with the redox-responsive behavior of polymeric nanocarriers reported elsewhere. 31,60The redoxresponsive behavior of the blank copolymer was further evaluated by estimating the molecular weight or elution time of the copolymer from APC after treatment with GSH.A concentration of 4 mg/mL of Bi(HOOC−PEG-PLGA)-S 2 was treated with 5 mM GSH overnight, and then its molecular weight was examined using APC.As depicted in Figure S5, the APC tracing graph showed a bimodal distribution with a halved molecular weight, indicating the cleavage of disulfide bonds with GSH.On the other hand, the control group (Bi(HOOC−PEG-PLGA)-S 2 in PBS) showed a unimodal peak with no change in its molecular weight.

Cellular Internalization of DOX-Loaded Micelles.
The cellular internalization of DOX@Bi(Dig−PEG-PLGA)-S 2 micelles was determined qualitatively by tracing the red fluorescent intensity of DOX in the cytosol and nuclei as compared to that of DAPI (a blue fluorescent dye used to stain nuclei) using a fluorescent microscope.The overlapping of red fluorescence and blue fluorescence in the microscopic cell imaging referred to the internalization of the DOX@Bi(Dig− PEG-PLGA)-S 2 micelles and subsequent delocalization of DOX to the nuclei. 61As shown in Figure 4b, the intensity of red fluorescence of cells treated with free DOX, DOX@ Bi(HOOC−PEG-PLGA)-S 2 , and DOX@Bi(Dig−PEG-PLGA)-S 2 , with an equivalent DOX dose of 5.5 μg/mL, was time-dependent.Cells treated for 12 h showed relatively more intensified red fluorescence than the corresponding cells treated for 4 h.As the incubation time increased, the cellular internalization and cytosolic redox pool-triggered release of DOX from the micelle and its subsequent translocation to the nuclei could increase.Interestingly, the intensity of red fluorescence of cells treated with DOX@Bi(Dig−PEG-PLGA)-S 2 was higher than that of DOX@Bi(HOOC−PEG-PLGA)-S 2 -treated cells, suggesting that the cellular trafficking of DOX@Bi(Dig−PEG-PLGA)-S 2 was enhanced by the targeting ability of the digoxin moiety.The plasma membrane of HeLa cells has an overexpressed Na + /K + ATPase that might enhance the cellular internalization of digoxin-modified micelles.Unlike DOX@Bi(HOOC−PEG-PLGA)-S 2 -treated cells, the red florescence intensity of DOX in HeLa cells incubated with DOX@Bi(Dig−PEG-PLGA)-S 2 was superimposed with the blue fluorescence intensity of DAPI, asserting that the release and translocation of DOX from  DOX@Bi(Dig−PEG-PLGA)-S 2 micelles to the nuclei was higher than DOX@Bi(HOOC−PEG-PLGA)-S 2 micelles.This important phenomenon reiterated the selective targeting ability of digoxin-modified micelles and the controlled release of DOX in the redox environment of cancer cells.The relatively smaller size of DOX@Bi(Dig−PEG-PLGA)-S 2 micelles than DOX@Bi(HOOC−PEG-PLGA)-S 2 micelles might also play a part in the enhanced crossing of the cell membrane.However, the red fluorescence intensity of cells treated with DOX@ Bi(Dig−PEG-PLGA)-S 2 was slightly weaker than that of cells treated with free DOX.The rapid and simple diffusion phenomenon of free DOX could possibly contribute to the abundant intracellular DOX levels, as manifested by the intensified red fluorescence in the nuclei of HeLa cells (treated with free DOX).Overall, the presence of digoxin as a targeting moiety and disulfide linkage as a redox-responsive spot had a profound effect on the cellular trafficking and accumulation of DOX@Bi(Dig−PEG-PLGA)-S 2 micelles in cancer cells.
3.8.Cytotoxicity of Blank and DOX-Loaded Micelles.The biocompatibility of copolymers used as nanocarriers is an important concern in DDSs. 62Therefore, the safety profile of the block copolymer, Bi(Dig−PEG-PLGA)-S 2 , was assessed in vitro using normal (NIH-3T3) and cancer (HeLa) cell lines prior to anticancer activity testing.As shown in Figure 4a, the MTT assay revealed that the viability of both cell lines (NIH-3T3 and HeLa cells) treated with Bi(Dig−PEG-PLGA)-S 2 for 24 h was remarkably high (≥80%), even at a maximum concentration of 200 μg/mL.The result reiterated that the Bi(Dig−PEG-PLGA)-S 2 copolymer is devoid of notable cytotoxic effects against normal and cancer cells, making it safe and convenient for drug delivery applications.Inspired by the aforementioned scenario, the in vitro anticancer activity of DOX@Bi(HOOC−PEG-PLGA)-S 2 and DOX@Bi(Dig−PEG- PLGA)-S 2 was studied against HeLa cells.Following a 24 h treatment of HeLa cells with free DOX (positive control), the DOX@Bi(HOOC−PEG-PLGA)-S 2 and DOX@Bi(Dig−PEG-PLGA)-S 2 in a dose-dependent manner (equivalent DOX concentration ranging from 0.5 to 12.5 μg/mL), MTT assay was performed.As depicted in Figure 4c, the viability of cells treated with DOX@Bi(Dig−PEG-PLGA)-S 2 was significantly lower (∼27%) than cells treated with DOX@Bi(HOOC− PEG-PLGA)-S 2 (∼38%) at a maximum dose of DOX (12.5 μg/mL) (p < 0.05).However, the viability of cells treated with DOX@Bi(Dig−PEG-PLGA)-S 2 was relatively comparable with that of cells treated with free DOX at equivalent doses.This might be attributed to the digoxin-directed enhancement of cellular internalization in cells treated with DOX@Bi(Dig− PEG-PLGA)-S 2 and the sensitization of HeLa cells toward DOX by digoxin. 63The result was in line with the cellular uptake of DOX@Bi(Dig−PEG-PLGA)-S 2 (Figure 4b), which was comparatively higher than that of DOX@Bi(HOOC− PEG-PLGA)-S 2 and obviously might trigger subsequent cellular cytotoxicity.The half maximal inhibitory concentration (IC 50 ) of DOX@Bi(Dig−PEG-PLGA)-S 2 , DOX@Bi-(HOOC−PEG-PLGA)-S 2 , and free DOX were 5.16 μg/mL, 6.91 μg/mL, and 4.08 μg/mL, respectively.The relatively higher IC 50 values of DOX@Bi(HOOC−PEG-PLGA)-S 2 and DOX@Bi(Dig−PEG-PLGA)-S 2 than the free DOX might be due to difference in the cellular trafficking mechanisms (diffusion versus receptor-mediated endocytosis) and relatively slow release rate of DOX from the micelles. 26,64.9.Apoptosis Assays of DOX-Loaded Micelles.To complement the MTT assay, an in vitro apoptotic assay was implemented in our study.The apoptosis of cells induced by DOX@Bi(Dig−PEG-PLGA)-S 2 in comparison to DOX@ Bi(HOOC−PEG-PLGA)-S 2 and free DOX against HeLa cells was studied through annexin V/PI double staining using confocal laser scanning microscopy (CLSM) imaging of apoptotic and necrotic cells after 12 h treatment with a corresponding DOX concentration of 5.5 μg/mL.As shown in Figure 5a, the intensity of green fluorescence was remarkably higher on cells treated with DOX@Bi(Dig−PEG-PLGA)-S 2 than DOX@Bi(HOOC−PEG-PLGA)-S 2 , signifying that more cells undergo apoptosis by the synergistic effect of DOX and digoxin.Moreover, the presence of digoxin on the surface of the DOX@Bi(Dig−PEG-PLGA)-S 2 micelles improved intracellular accumulation through digoxin-receptor-mediated endocytosis and concurrent release of DOX in the cytosol of HeLa cells, followed by a substantial cellular apoptosis. 65,66.10.Flow Cytometry Analysis of DOX-Loaded Micelles.To further investigate the mechanisms involved in the therapeutic efficacy of DOX@Bi(Dig−PEG-PLGA)-S 2 against HeLa cells, flow cytometry analysis was performed, including free DOX and DOX@Bi(HOOC−PEG-PLGA)-S 2 for comparison.After 12 h of incubation with free DOX, DOX@Bi(HOOC−PEG-PLGA)-S 2 , and DOX@Bi(Dig− PEG-PLGA)-S 2 at an equivalent DOX concentration of 5.5 μg/mL, HeLa cells were double stained with PI and annexin V-Alexa Flour 488, and the apoptotic and necrotic cells were quantified in terms of percentage.As depicted in Figure 5b, the percentage of HeLa cells undergoing early/late apoptosis was notably higher for cells treated with DOX@Bi(Dig−PEG-PLGA)-S 2 compared to nontreated cells (negative control) or cells treated with DOX@Bi(HOOC−PEG-PLGA)-S 2 under normal physiological conditions (37 °C and pH 7.4).The total percentage of early/late apoptotic cells in the DOX@Bi(Dig− PEG-PLGA)-S 2 -treated group was 67% (19.9% early apoptotic cells and 47.1% late apoptotic cells), which was higher than the total apoptotic cells of the DOX@Bi(HOOC−PEG-PLGA)-S 2 -treated group (34.2% early apoptotic cells and 18.9% late apoptotic cells).This suggested that the targeting moiety, digoxin, in DOX@Bi(Dig−PEG-PLGA)-S 2 most likely increased cellular internalization of DOX@Bi(Dig−PEG-PLGA)-S 2 and the subsequent cleavage and/or oxidation of S−S bonds in the micelles due to high levels of redox pool in the cytoplasm of cancer cells, resulting in swelling or disassembly of micelles and prompt release of DOX in the cells to induce apoptosis.Moreover, the anticancer and chemosensitizing effects of digoxin may have resulted in synergistic apoptotic effect on HeLa cells. 67These results align with the data obtained from CLSM imaging and the MTT assay (Figure 4b,c).On the other hand, unlike positive control cells (free DOX-treated cells) with a relatively higher percentage of early/ late apoptotic cells (63.6%) and necrotic cells (14.5%), the total number of early/late apoptotic cells in the negative control cells (nontreated cells) was insignificant (3.98%).Overall, this study revealed that Bi(Dig−PEG-PLGA)-S 2 micelles could serve as multifunctional nanocarriers that could potentially improve the efficacy and safety of chemotherapeutic drugs owing to the cancer cell recognition ability and synergistic chemotherapeutic effects for cancer treatment.

CONCLUSION
In conclusion, this study features the potential of redoxresponsive Bi(Dig−PEG-PLGA)-S2 micelles as an effective drug delivery system for cancer treatment.These micelles, synthesized from an amphiphilic block copolymer, reveal selective accumulation in the tumor tissues via receptormediated endocytosis and respond to the unique redox environment of the tumor tissue.Moreover, the integration of digoxin improves the therapeutic efficiency of DOX, leading to rapid apoptosis of cancer cells.With desirable drug loading capacity and controlled release of DOX triggered by the cancer cell redox pool, Bi(Dig−PEG-PLGA)-S2 micelles significantly improve the cellular accumulation, temporal release, and subsequent cytotoxicity of DOX in cancer cells.Overall, our findings revealed that Bi(Dig−PEG-PLGA)-S2 micelles showed a promising drug delivery system for cancer therapy.

2 . 11 .
In Vitro Cytotoxicity of the Blank and DOX-Loaded Micelles.The biocompatibility of Bi(Dig−PEG-PLGA)-S 2 was studied in normal cell lines (NIH-3T3 cells and

Table 1 .
Molecular Weight of the Synthesized Copolymer Measured by APC a copolymersMn (g/mol) Mw (g/mol) Mp (g/mol) Đ a Mn: number average molecular weight; Mw: weight average molecular weight; Mp: molecular weight at peak maximum.

Table 2 .
Size and Surface Charge of the Micelles were Determined from DLS Measurements Taipei 10607, Taiwan; Advanced Membrane Materials Center, National Taiwan University of Science and Technology, Taipei 10607, Taiwan; R&D Center for Membrane Technology, Chung Yuan University, Chung-Li 320, Taiwan; orcid.org/0000-0002-7034-6205;Email: h.c.tsai@mail.ntust.edu.twSzu-Yuan Wu − Department of Food Nutrition and Health Biotechnology, College of Medical and Health Science and Department of Healthcare Administration, College of Medical and Health Science, Asia University, Taichung 413, Taiwan; Big Data Center, Lo-Hsu Medical Foundation, Division of Radiation Oncology, Department of Medicine, Lo-Hsu Medical Foundation, and Cancer Center, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan 256, Taiwan; Graduate Institute of Business Administration, Fu Jen Catholic University, Taipei 242, Taiwan; Centers for Regional Anesthesia and Pain Medicine, Taipei Municipal Wan Fang Hospital, Taipei Medical University, Taipei 110, Taiwan; Email: szuyuanwu5399@gmail.com Corresponding AuthorsHsieh-Chih Tsai − Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology,